A green compliant hand-held selective electrode device for monitoring active pharmaceuticals and the kinetics of their degradation

An in-line smartphone connected to a screen-printed selective electrode hand-held device was used to determine the concentration of distigmine bromide (DB) in its pure and dosage forms as well as its degradation kinetics by continuously measuring the change in the produced emf over time. The main objective, supported by the data presented, is to produce a highly reliable smartphone integrated selective sensor as a portable analyzer with potential high cloud connectivity combining a wide linear dynamic range, the fastest response time with the lowest limits of detection and quantitation while best integrating green analytical chemistry principles. The choice of ionophore used in this approach was guided by computation and the data obtained was compared with traditional analytical techniques. DB, for which there are no previously reported stability-indicating methods and for which four novel such methods are proposed here, was selected as a model drug for this work. At-line UV-spectrophotometry DB assay was obtained by measuring the difference between the spectra of the degradation product and the same concentration of intact drug. The degradation kinetics were studied by this method through tracking the decrease of DB absorbance and/or the increase of a generated degradation product signal over time. Off-line separation based HPLC and TLC stability-indicating methods for DB were also presented. All methods employed in this work were validated for accuracy, precision, specificity, repeatability, linearity, range, detection and quantification limits according to the ICH guidelines and were applied to the analysis of laboratory prepared mixtures as well as commercial products. While all methods proposed were shown to be highly reliable, the smartphone integrated selective sensor is highlighted as a portable analyzer with potential high cloud connectivity and was shown to combine a wide linear dynamic range, the fastest response time with the lowest limits of detection and quantitation while best integrating green analytical chemistry principles.


Results and discussion
Methods development. ISE membrane optimization. To improve the selectivity and LOD of the developed sensor 43,44 , the ion-selective membrane responsible for the sensing mechanism was impregnated with a lipophilic ion-exchanger and an ionophore. The former in this case was potassium tetrakis 4-chlorophenyl borate (KTCPB) that selectively includes distigmine di-cations (DG) in the organic layer and excludes the counter ions into the bulk aqueous phase, resulting in interfacial charge separation. Strong intermolecular association and complexation energies between the selected ionophore and DG as well as a presence of a fourfold molar excess of the ionophore relative to DG would reduce the concentration of free DG and thus, in turn lowers the rate of DG transfer across the membrane. This strong intermolecular association may also allow the DG within the complex with the chosen ionophore to exhibit a higher lipophilic character than it would as a free molecule. This would also lower transmembrane DG flux, thus improve signal stability, sensitivity and increase the life span of the membrane sensor produced. Two potential molecular receptors and ionophores for the DG were consid- www.nature.com/scientificreports/ ered, these were non-hydroxylated calixarene 6, NCX6 and hydroxylated calixarene 6, CX6, the latter we have previously shown to be successfully used as an ionophore for the determination of singly positive choline ions in milk 18 . DFT calculations were employed to elucidate the structure of DG, the structures of both ionophores, as well as structures of their monomeric and dimeric complexes with DG. Calculations were also employed to detail the types of binding involved, provide values binding energies as well as to calculate the logP values of the free species and the associated complexes.
Ionophore selection. The calculated most stable conformations of the two potential ionophores NCX6 and CX6 as well as their molecular electrostatic potential surfaces are shown in Figure S1 of the supplementary material. Both NCX6 and CX6 are characterized by cavities that are conical and non-symmetric. This cavity is stabilized in the case of CX6 by five hydrogen bonds between hydrogen atoms and phenolic oxygen atoms as detailed in Figure S1. The most stable conformation of DG and the corresponding molecular electrostatic potential surface clearly showing the two electron deficient sites on either end of the extended DG molecule are shown in Panels A and B in Figure S2 of the supplementary material, respectively. Figure S3 of the supplementary material, shows the most stable 1:1 DG-NCX6 and DG-CX6 complexes and their corresponding molecular electrostatic potential surfaces. In either case one of the cationic pyridinium rings of DG is being hosted within the cavities of NCX6 or CX6. This lead to the delocalization of the positive charge of the complexed pyridinium rings as is evident in panels C and D of Figure S3 as well as the expansion and deformation of the cavities in these molecular hosts. In the case of CX6, this cavity expansion is also accompanied by the reduction in the number and the elongation of the remaining intramolecular hydrogen bonds. Both the 1:1 DG-NCX6 and DG-CX6 complexes are stabilized by cation-π interactions between the pyridinium tertiary nitrogen atoms acting as a π acceptors and the surrounding π cloud of the calixarene aromatic rings acting as the π donors. These interactions are evidenced through a comparison of the plotted non-covalent interactions and the reduced gradient plots in Figures S4 and S5. The calculated Gibbs binding free energies of complexation in a 1-nitro propane medium for the 1:1 DG-NCX6 and DG-CX6 species were determined to be − 15.8 and − 13.6 kcal mol −1 respectively. These values obtained in a 1-nitro propane medium which closely approximates the dielectric constant of 2-nitro phenyl octyl ether, the medium used experimentally to dissolve the ionophores, show that both of these complexes are stable and relatively close in binding free energy with a difference of 2.2 kcal mol −1 . However, since a fourfold molar excess of the chosen ionophore relative to DG would be used in order to minimize the concentration of free DG and thus, in turn lowers the rate of DG transfer across the membrane calculations on 1:2 DG-NCX6 and DG-CX6 complexes were performed. These calculations show that in both cases, the lowest conformation of the 1:2 complexes formed, have the two cationic pyridinium rings of DG, each inserted in the cavity of a separate host to produce near symmetrical host-gest complexes. The geometries for the 1:2 DG-NCX6 and DG-CX6 complexes are highlighted in Panels A and B respectively in Fig. 2. The corresponding molecular electrostatic potential surfaces presented in Panels C and D in Fig. 2; on the other hand, show the distribution of the positive charge over the structure as a result of host-guest complexation. The  A and B) show optimized geometrical structures for 2:1 DG complexes with NCX6 and CX6 respectively as obtained using the B97D/6-31G(d,p) level of theory (6-31 + G(d,p) was used for oxygen atoms). Panels (C and D) show molecular electrostatic potential surfaces for the DG complexes with NCX6 and CX6 respectively as obtained from the gas phase optimized structures using cubegen utility in the Gaussian 16 package. The color code indicates electron rich (red) and electron deficient sites (blue). www.nature.com/scientificreports/ non-covalent character of the interactions in these 1:2 DG complexes with either NCX6 and CX6 are clearly shown in Figure S6 of the supplementary materials. Figure S6 shows in both complexes, each of the pyridinium rings to be surrounded by six aromatic rings from different directions, thus these cation-π interaction can be identified in different binding fashions of edge to face and face to face. These interactions are significant as supported by the calculated Gibbs binding free energy for the 1:2 DG-NCX6 and DG-CX6 complexes obtained in a 1-nitro propane medium to be − 30.67 and − 30.24 kcal mol −1 respectively. In general, cation-π interactions ranging from − 12 to − 65 kcal mol −1 are reported 45 . This further indicates that these non-covalent guest-host interactions play a significant role in stabilizing the resulting complexes and play a part in reducing the concentration of free DG and thus, in turn inhibits the rate of DG transfer across the membrane. These 1:2 DG-NCX6 and DG-CX6 complexes also exhibited a significantly higher lipophilic character than that of free DG as demonstrated by the calculated logP values of 9.98 and 7.24 for the two complexes respectively relative to − 3.55 for DG. This also is expected to lower transmembrane DB flux thus improving the signal stability, sensitivity and life span of the membrane sensor produced and clearly demonstrates the suitability of both ionophores. In this work we have adopted CX6 as the ionophore of choice due to its commercial availability and lower cost.
In-line smartphone-based ISE measurements. The potentiometric time traces obtained by measuring the emf in solutions of DB employing six different screen-printed ion-selective electrode sensors connected to a smartphone (n = 6) are shown in Fig. 3 as plots of the emf values obtained against the logarithmic concentrations of DB with a LOD of 78.0 nM. The average response time for the sensors employed was less than 10 s, which allowed for rapid emf recordings and near real time response during the kinetic monitoring of DB hydrolytic degradation. The measurements were obtained by stepwise dilution of the DB solution, at a 10 mM initial concentration, that was performed by removing an aliquot of the DB solution and its replacement with an equal volume of Britton-Robinson buffer (BRB) at pH 7.0 under continuous emf measurements. A typical calibration curve as a function of DB concentration obtained from the average of the six-potentiometric time traces is presented in Figure S7 in Supplementary Materials. This shows a Nernstian linear dynamic range of several orders of magnitude with a R 2 = 0.9986 for the linear correlation, an average slope value of 27.51 ± 0.72 mV dec −1 and a standard potential, E 0 , of 305.44 ± 1.45 mV. The pH effect on the potentiometric measurements was studied by www.nature.com/scientificreports/ calibrating the sensor at pH values in the range of 2-13 by using BRB. These results presented in Fig. 4 show no significant difference in the corresponding Nernstian slopes within the pH range 3-8. Figure 4 also shows that at pH values ≥ 9, non-Nernstian slopes were obtained which is attributed to the degradation of the drug. It was demonstrated with different tests using separate solutions that the sensors exhibited greater selectivity towards DB relative to its degradation product THMP. This may be due to the higher lipophilicity of DB compared to THMP at the working pH, where log K pot DB.THMP = − 2.50. This discrimination towards DB over its degradation product THMP is a factor in allowing accurate monitoring of DB during the initial stages of its chemical catalyzed hydrolysis degradation reaction which were studied by continuous recording of the emf values for DB solutions at pH values 10 and 11 each at thermostatically controlled temperatures of 25, 30 and 35 °C all ± 2 °C and at an initial DB concentration of 0.1 mM.
At-line spectrophotometric measurements. DB degrades to give THMP, which in turns undergoes changes in its spectral profile as shown in Fig. 5. These pH dependent spectral profile changes are most likely  www.nature.com/scientificreports/ due to the reversible ionization of the phenolic hydroxyl group conjugated with the pyridine ring. Figure 5 shows the appearance of a THMP absorbance peak at λ max = 320 nm at pH 4 and 6. This is a spectral region where no absorption of the intact drug DB occurs as shown in Fig. 6 where an overlay of DB and THMP spectra both at pH 7 are presented. The proposed method depends on measuring the difference between the spectrum of THMP obtained from the hydrolysis of DB and the spectrum of the same concentration of the intact drug. In this way, any signal due to amount of the degradation products present in the DB samples was subtracted from the readings at the corresponding wavelength after hydrolysis and the difference in absorbance before and after hydrolysis would correspond only to the intact drug. Calibration is then obtained by accurately transferring volumes of DB equivalent to 100.0-600.0 μg into a series of test tubes, each containing 1.0 mL 0.05 M NaOH, and were kept in a water bath at 100 °C for 10 min. The contents of each test tube were neutralized using 0.05 M HCl to pH 7.0 and transferred quantitatively in separate 10 mL volumetric flasks which were subsequently completed to the mark with BRB at pH 7. The absorbance of each solution was then recorded at 320 nm against a corresponding blank solution containing the same concentration of intact DB in BRB at pH 7. Figure S8 of the Supplementary Material shows the results of applying this suggested procedure to obtain differences between the spectra at λ = 320 nm for concentrations of 10.0-60.0 μg/mL for the DB degradation product, THMP, against the same respective concentrations of intact DB as blank. This procedure has also produced a strong linear response with a correlation coefficient R 2 = 0.9999 as shown in Figure S9 of the Supplementary Material. The hydrolytic degradation of DB under the same conditions to produce THMP as previously described above was investigated. Complete hydrolytic degradation of DB to produce THMP was achieved at several concentrations of DB by accurately measuring 100-600 μg volumes of DB which were transferred separately into a series of test tubes. To each test tube, 1.0 mL of 0.05 M NaOH was added then kept in a water bath at 100 °C for 150 min. Separately, the contents of each test tube were transferred quantitatively into six 10 mL volumetric flasks which were subsequently completed to the mark with BRB. In each sample THMP absorbance signal was recorded at 320 nm, as this wavelength minimized interfering signal from DB as shown in Fig. 6, against the corresponding blank solution containing the same concentration of intact DB dissolved in BRB at pH 7 allowed for the construction of a THMP calibration curve. DB degradation in two BRB solutions of pH 10 and 11 thermostatically controlled at 25, 30 and 35 °C all ± 2 °C were investigated. This was accomplished by scanning the DB degradation reaction every 3 min from the start of the reaction over the 150 min time span at the respective controlled temperatures through the increase of the THMP signal at 320 nm that also corresponds to the decrease of DB signal at 270 nm with increasing time as shown in Fig. 7.

Off-line chromatographic measurements.
In HPLC measurements, sodium-1-heptane sulphonate was used as an ion-pairing agent in order to allow for the complete separation between DB and its THMP and to improve peak symmetry and sharpness as well as reduce peak tailing of the drug. The amount of acetonitrile used was also adjusted, since at higher acetonitrile concentration, separation occurred but with excessive tailing and increased retention time of the DB peak. Using a 0.01 M sodium-1-heptane sulphonate:acetonitrile (70:30 v/v) mobile phase and the chromatographic conditions previously described, a retention time of 4.45 ± 0.02 min for DB and 1.91 ± 0.02 min for its alkaline degradation product THMP was achieved with excellent peak shapes as shown in Fig. 8. Using the external standard approach for calibration, a linear correlation R 2 = 0.9996 was www.nature.com/scientificreports/ obtained between the relative peak area and the corresponding drug concentrations in the range 4-32 µg/mL at λ = 220 nm as shown in Figure S10 of the Supplementary Material. For TLC densitometry, many experiments were conducted to achieve optimum separation of the drug and its degradation product. The trials were focused on changing the composition of the developing system by varying the percentages of organic solvents. These systems included methanol : ethyl acetate : NH 3 (4:1:0.2, by volume); acetone:butanone:H 2 O:NH 3 (5:3:2:0.01, by volume) and acetone:butanone:acetic acid:H 2 O:NH 3 (1:1:1:1:0.03, by volume). The first developing system showed poor resolution for the two analytes with peak tailing. The second system was not suitable for DB, which remained almost on the spotting line. However, upon adding acetic acid to the developing system, the obtained TLC chromatogram was promising. After fine adjustment of the ratios and addition of acetic acid, the optimum developing system was acetone:butanone:acetic acid:H 2 O:NH 3 (1:1:1:1:0.03, by volume). Figure 9 shows sharp symmetric peaks with good resolution for the two components with Rf values 0.31 and 0.64 for DB and THMP, respectively. This separation allowed for the scanning of DB  Off-line monitoring of the hydrolytic degradation of DB using HPLC and TLC were performed. In both cases an aliquot of the DB degradation reaction was removed every 3 min from the start of the reaction and for 100 min. These aliquots were neutralized to pH 7 with BRB and 20 µL were injected in the HPLC using the same chromatographic conditions employed for the assay measurements of DB as described earlier. These conditions produced complete separation and excellent peak shapes for DB and THMP, whose retention times were verified by separate injections of each of these compounds under the same chromatographic conditions. In the case of TLC, the aliquots removed from the DB degradation reaction, were spotted after their neutralization onto silica gel 60 F 254 TLC plate employing the same TLC chromatographic conditions previously described. The intensities of the peaks of the analyte of interest were measured and relative concentration profiles were determined from the relative peak areas versus reaction times.
Validation of developed methods. All the methods employed in this work were validated for accuracy, precision, specificity, repeatability, linearity, range, detection and quantification limits according to the ICH guidelines 46 Table 1 which also summarizes several metrological parameters of the proposed sensor showing the suitability of all of the developed methods for their proposed use as evaluated with respect to IUPAC recommendations 47 . Table 1 also shows that the proposed ISE method possesses one of the widest linear ranges and the lowest LOD and LOQ values of all the methods employed here. The proposed methods were also employed successfully for assaying DB in Ubretid® tablets. The reliability and accuracy of recoveries of all the proposed methods as listed in Table 2 relative to the official method for DB assay clearly show that the proposed methods are unaffected by excipients and other non-active components present in commercial formulations.
Degradation kinetics. The analytical methods employed here are all stability indicating and were employed for monitoring the degradation kinetics of DB at pH 10 and 11 in each case at 25, 30 and 35 °C all ± 2 °C. For in-line ISE measurements, the rapid response time for DB of less than 10 s offered multiple advantages. This allowed for near instantaneous emf recording and thus near real time observation of the hydrolysis behaviour of DB. This also allowed for a significantly larger number of data points to be obtained relative to the at-line and off-line methods employed here which increases the confidence in constants determined for the kinetics of DB hydrolysis based on data obtained through this in-line approach. The near instantaneous response time of the sensor also facilitated the recording of the emf at pH 10 and 11 before the start of the DB hydrolysis process. This in turn allowed for calibration curves obtained at these pH values employing this methodology to show no statistically significant differences in their linearity or Nernstian slopes relative to those obtained by the same method at pH 7.
At-line spectrophotometric measurements were conducted to investigate DB degradation at the same pH and temperature conditions mentioned above. Here, complete hydrolytic degradation of DB to produce THMP was achieved at several concentrations of DB by accurately measuring volumes of DB, equivalent to 100-600 μg, which were transferred separately into a series of test tubes. To each test tube, 1.0 mL of 0.05 M NaOH was added then kept in a water bath at 100 °C for 150 min. Separately, the contents of each test tube were neutralized using 0.05 M HCl to pH 7.0 and transferred quantitatively into six 10 mL volumetric flasks which were subsequently www.nature.com/scientificreports/ completed to the mark with BRB (pH 7). In each sample THMP absorbance signal was recorded at 320 nm, as this wavelength eliminated interfering signal from DB against the corresponding blank solution containing the same concentration of intact DB dissolved in BRB at pH 7 allowing for the construction of a calibration curve. The DB hydrolytic degradation studies were performed by auto scanning the reaction every 3 min over a period of 3.5 h. These conventional scans are shown in Fig. 7 where the DB peak at 270 nm gradually decreased and the THMP peaks at 252 and 320 nm increased over time. Figure 7 and more visibly in Movie S12 of the Supplementary Material show two isosbestic points at 260 and 287 nm that indicate the absence of side reactions. The kinetic degradation of DB can thus be expressed either directly through the decrease of DB signal at 270 nm or through the increase of THMP signal at 320 nm with time. Both these approaches consistently correlated fairly well with that obtained by ISE.
Off-line experiments were performed for monitoring the degradation of DB by placing a fixed concentration in BRB at pH 10.0 and 11 all at 25 ± 2 °C where aliquots were withdrawn at 5 min intervals. These aliquots were neutralized to pH 7 and completed to a definite volume, and subsequently spotted on the silica gel 60 F 254 TLC plates and injected in the HPLC using the chromatographic conditions described previously for each technique. For both methods, relative decreasing concentration profiles of DB were determined versus reaction times.
For all of the approaches employed in this work the logarithm of the DB concentration was then plotted with respect to time for measurements at pH 10 and 11 at each of the three temperatures employed as shown in Fig. 10 in the case of the smart phone integrated potentiometric sensor. These plots all show pseudo-first-order degradation of DB through its hydrolysis in a large excess of BRB. From these plots, degradation rate constants and the corresponding half-lives of DB, t 1/2 were determined and are all shown to be in good agreement across all methods employed as summarized in Table 3. This data also shows the hydrolysis rate of DB increasing and its half-life, t 1/2 , decreasing with increasing temperature and pH. The activation energy, E a , for the degradation reaction was calculated using Eq. 1 48 .   Evaluation of the greenness profile of the proposed methods. In this work, all proposed methods were evaluated and ranked in light of their adherence to green analytical chemistry principles 1 according to   42 . The GAPI assessment tool serves as a valuable semi-quantitative technique for assessing the overall greenness of an analytical technique. This approach expands on previous assessment tools through rating analytical methods on various aspects including sample preparation, collection, safety, potential health impact of the substances used as well as waste produced. GAPI uses a three-color scale for each sector, being green to yellow to red, similar to traffic lights, where green denotes a benign technique and red denotes a non-environmentally friendly one while yellow is somewhere in the middle. The GAPI pictograms obtained upon employing this system on all our proposed techniques, as shown in Table 4, demonstrates that the smart phone integrated ISE method was the greenest approach with green in 13 sectors, while the spectrophotometric, HPLC and TLC methods had six, three and four sectors green respectively. The ESA was applied for a more comprehensive assessment of the greenness profile of the proposed methods. This scale assigns penalty points to each non-green procedural component, which are then subtracted from a base of 100 to provide a final numerical score. The higher the score the greener is the analytical process. Table 4 lists these scores, as computed for the proposed methods in this work, showing the smartphone based electrochemical sensor approach to be the most green followed by spectrophotometry, HPLC and TLC. This scale has the advantage of being semi-quantitative, with numerical values as a reference for assessing greenness. However, there are also many disadvantages to using this scale. These include not providing information about the type of hazards or the factors that contribute to the negative environmental impact of an analytical procedure, all information that is most useful in the development of a better eco-friendly method.
For this reason a third approach, AGREE, which is an all-encompassing, straightforward and flexible assessment tool that provides interpretable and instructive results, that was recently introduced, was employed here 47 . In this approach, the assessment criteria were derived from the 12 principles of GAC. The tool output is a clocklike graph, with color representation similar to the GAPI system but with an overall score displayed in the middle. One of the advantage of this metric is the availability of open-source, downloadable software (https:// mostw iedzy. pl/ AGREE), which simplifies its application. This assessment tool also indicates that the ISE methodology employed in this work possessed the highest score, 0.88, and highest number of green segments, followed by UV-VIS, HPLC and TLC with scores of 0.68, 0.51 and 0.45 respectively as listed in Table 4.
In addition to its greenness profile, the smartphone-based ISE technique offers several unique advantages. These include its fast response time providing the ability for continuous real time reaction monitoring as discussed earlier; its compatibility with microfabrication processes; its portability and miniaturization permitting its use for in-situ measurements; its affordability and ease of use; its high reproducibility at a wide linear range as well as its nano-molar level LOD. These unique advantages suggest the suitability of this method for in vitro kinetic testing in possible conjunction with ingestible electronics.

Conclusion
Four novel stability-indicating methods for DB were reported and tested on commercial formulations. This represents a major advancement for the analysis of DB for which no stability-indicating method was previously reported. The methods described here were an in-line method based on a smartphone connected to a screen-printed selective electrode hand-held device, an at-line UV-spectrophotometric strategy and two off-line www.nature.com/scientificreports/ strategies, utilizing HPLC and TLC methods. All the methods were validated for accuracy, precision, specificity, repeatability, linearity, range, detection and quantification limits according to the ICH guidelines and were applied to the analysis of laboratory prepared mixtures as well as formulated commercial products. The analytical methods employed here were applied for monitoring the degradation kinetics of DB at pH 10 and 11 in each case at 25, 30 and 35 °C all ± 2 °C. For all of the methods of pharmaceutical analysis used and for all measurement conditions, the logarithms of the DB concentrations were plotted with respect to time. All plots showed pseudofirst-order degradation of DB through its hydrolysis in a large excess of BRB. From these plots, degradation rate constants and the corresponding half-lives of DB, t 1/2 were determined and are all shown to be in good agreement across all methods employed. The activation energy for the degradation reaction was estimated to be 9.90 ± 0.97, 10.67 ± 1.80 and 10.02 ± 2.15 kcal mol −1 as obtained by ISE, spectrophotometry and HPLC respectively. While all stability-indicating methods proposed were shown to be highly reliable, the smartphone integrated selective sensor is highlighted as a portable analyzer with potential high cloud connectivity and was shown to combine a wide linear dynamic range, the fastest response time with the lowest limits of detection and quantitation while best integrating green analytical chemistry principles. At-line spectrophotometric measurements. Spectrophotometric at-line absorption measurements employed a UV-Vis double beam PC 8 scanning auto cell spectrophotometer, UVD 3200 (Labomed, INC, USA). DB determination was achieved by measuring the difference between the spectrum of the alkaline degradation product and the spectrum of the same concentration of the intact drug.

Methods
Off-line chromatographic measurements. The first off-line technique employed in this work made use of an Agilent HPLC system equipped with a G1310A isocratic pump, a G1314 variable wavelength detector and a Rheodyne injector (model 7725I). The HPLC measurements were performed on 20 µL samples using a 5 μm C 18 Zorbax TM analytical column (25 cm × 0.46 cm) and a mobile phase of 0.01 M sodium-1-heptane sulphonate : acetonitrile (70:30 v/v) at a flow rate of 1.0 mL min −1 and 220 nm UV-detection. The sample and the mobile phase were filtered by 0.22 μm and 0.45 μm Millipore membrane filters, respectively. The mobile phase was then degassed in an ultrasonic bath for 15.0 min immediately before use. Aliquots of DB equivalent to 40.0-320.0 μg were accurately transferred into 10 mL volumetric flasks and the volume was completed with mobile phase. Calibration curves were then constructed for DB by plotting the relative peak areas of DB as a function of respective concentrations.
The second off-line technique selected in this work was densitometry. The device employed was equipped with a UV lamp, a Camag Linomat 5 autosampler with a 100 µL micro-syringe, a TLC scanner-Model 3 S/N 130319 and winCats software for densitometric evaluation (CAMAG, Muttenz, Switzerland). The measurements were obtained using 20 cm × 20 cm TLC plates pre-coated with a 0.25 mm thick layer of silica gel 60 F 254 (E. Merck, Darmstadt, Germany). The solutions examined were applied as separate spots 20 mm from the bottom of the plates each with a 2 mm band length and developed at 25 ± 2 °C in the absorbance mode at 270 nm running a developing system of acetone:butanone:acetic acid:H 2 O:NH 3 (1:1:1:1:0.03 by volume). The drug solutions examined were applied as separate compact spots 15 mm from the bottom of the plates each with a 3 mm band length which were placed in a chromatographic tank saturated with the mobile phase for 30 min prior to development. The normal phase TLC-plates were developed over 8 cm in an ascending manner then left to dry in air and then were scanned at 220 nm. In order to calibrate this method, accurately measured aliquots equivalent to 2.0-12.0 μg DB were spotted on the TLC plates, using the Camag Linomat autosampler using the same TLC Computational details. Molecular orbital calculations for CX6, distigmine in its non-brominated form (DG) and their host-guest complexes were performed in the gas phase at the DFT level of theory using the Gaussian16 code 50 . All geometries were optimized without any constraints using the B97D exchange and correlation functional with the 6-31G(d,p) basis set for all atoms except for oxygen, for which the 6-31 + G(d,p) basis set which includes a diffuse function was employed. A similar computational protocol was reported to be successful for similar systems 18,51 . All critical points were characterized as local minima by means of harmonic vibrational analysis at the same level of theory by ensuring the absence of negative or imaginary frequencies. These frequency calculations were also used to calculate the Gibbs free energies for all species calculated. To account for solvent effects, the implicit polarizable continuum model (PCM) 52 was employed where a single point calculation using the gas phase optimized geometries were computed with the standard Pople type 6-311 + + g(d,p) basis set for all the atoms to calculate the solvation free energies. Since the medium employed experimentally, 2-nitro phenyl octyl ether, having a dielectric constant ε ¼ 23-24 53-56 is unavailable as a solvent in the Gaussian code, the solvent 1-nitro propane, having a dielectric constant of ε ¼ 23.7 was employed in these PCM calculations. The Gibbs free energies in solution were calculated for CX6, DG and complex between them as the sum of the gas-phase free energy G • gas and the solvation free energy G • solv , as previously reported 18,20,51 . The binding Gibbs free energy is then calculated using G • solv values as where G • CXDG , G • CX , G • DG are the G • solv of the CX6-DG complex, CX6 and DG respectively. To examine the hydrophobicity/hydrophilicity of the CX6-DG complex relative to each of CX6 and DG we have calculated the logP values per the protocol suggested by Nedyalkova et al. 57 . Briefly, full geometric optimization without constrains were performed for all systems under study using the SMD model employing the 6-31G(d,p) basis set for all atoms except for oxygen where the 6-31 + G9d,p) basis set was used. The SMD model was shown to be suitably applied, as a solvation model, to charged and neutral species alike 58 . In this model the solvation free energy is divided into bulk electrostatic and cavity dispersion contributions. The octanol/water partition coefficient, was obtained by using the SMD free energies obtained in these two solvents at 289.15 K which were applied to calculate the standard free energy associated with the transfer of the solute from water to octanol, �G • O/W , as shown below; The octanol/water partition coefficient was subsequently calculated according to The noncovalent interactions (NCI) between DG and CX6 in their complex geometries were calculated by means of reduced density gradients. These gradients were analysed using the Multiwfn software for the generation of an NCI surface where the reduced density gradients (RDG) were plotted using the VMD software employing an isosurface value of 0.3. Subsequently, plots of RDG and sign(λ 2 )ρ values with a RDG and density cutoff of 2.0 and 0.05 a.u respectively were produced to inform about the nature and strength of the NCIs. Molecular electrostatic potential surfaces were created using cuebgen utility in the Gaussian 16 package.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.